Development of an in vitro TurboID labeling assay for the detection of protoRAG-interacting proteins in the amphioxus

Abstract

The recombination-activating gene (RAG)-mediated V(D)J rearrangement is essential for adaptive immunity in jawed vertebrates. RAG evolved from an invertebrate RAG-like (RAGL) transposase, with the amphioxus protoRAG (BbRAG1L/BbRAG2L) serving as a key model. However, the regulatory mechanisms of protoRAG remain unclear. Here, we developed an in vitro proximity labeling assay using TurboID fused to BbRAG1L or BbRAG2L to identify interacting proteins from amphioxus hepatic cecum and colon lysates. The fusion proteins were expressed in Expi293F cells, purified, and incubated with amphioxus lysates in the presence of biotin and ATP. Biotinylated proteins were enriched via streptavidin beads and analyzed by LC-MS/MS. This approach enables the identification of protoRAG-interacting proteins without the need for transgenic amphioxus, providing a valuable tool to study the evolution of RAG regulation.

1. Introduction

The recombination-activating gene (RAG)-mediated V(D)J rearrangement mechanism is essential for generating the diversity of the adaptive immune system in jawed vertebrates. The RAG1 and RAG2 proteins form a tetrameric complex that recognizes and cleaves pairs of recombination signal sequences (RSSs). In jawed vertebrates, the N-terminal region of RAG1 and the C-terminal region of RAG2 are critical for the regulation of physiological function and enhancement of recombination accuracy. Moreover, the intracellular activity of RAG1/2 is rigorously regulated and is thought to represent the aspect shaped by host coevolution. However, these regulatory mechanisms remain incompletely understood, impeding our deeper understanding of the physiological functions of RAG.

Previous studies have shown that jawed vertebrate RAG evolved from invertebrate RAG-like (RAGL) transposases and underwent key structural changes during evolution. RAGL genes have been identified in multiple invertebrate species, but from Branchiostoma belcheri (Bb), the RAG-like protein in Bb amphioxus (BbRAG1L) and the RAG2-like protein in Bb amphioxus (BbRAG2L) are among the most extensively characterized to date. Lancelets (amphioxus) represent the most basal extant chordates (cephalochordates) that diverged from the other two chordate lineages (urochordates and vertebrates). Consequently, amphioxus has been considered a “living fossil” for studying the origins and evolution of adaptive immunity. Homologs of the core structural domain of RAG1 were discovered in the amphioxus genome in 2005, and the homologs were subsequently shown to exhibit recombinase activity in cooperation with murine-derived RAG2. A large number of transposon genes were then identified in the amphioxus genome, followed by the identification of target site duplication (TSD) and terminal inverted repeat (TIR) sequences in the amphioxus genome. Notably, BbRAG2L lacks the C-terminal region present in murine RAG2, including the acidic hinge and PHD domain. BbRAG1L coupled with BbRAG2L displays significant transposition activity in vitro and in human cell lines. In contrast, vertebrate RAG exhibits limited transposon activity in vitro and extremely low transposon activity in vivo. This leads to the question of how RAG lost its transposase activity during evolution. Structural comparison and functional experiments of BbRAGL (protoRAG) and vertebrate RAG have revealed that vertebrates undergo essential adaptive changes to eliminate the primitive transposase activity of protoRAG. Research on BbRAGL has therefore provided strong support for the hypothesis that RAG originated from a domesticated transposon.